1. Area of the Art
The invention relates generally to activated dyes and specifically to activated cyanine dyes with an acyl fluoride activating group, their synthesis and methods of use in labeling of biopolymers.
2. Description of the Prior Art
Many procedures employed in biomedical research and recombinant DNA technology rely heavily on the use of nucleotide or polynucleotide derivatives radioactively labeled with isotopes. However, the rapidly increasing costs of radioactive waste disposal, together with an increased awareness of the potentially harmful effects of exposure to radiation, have contributed to shifting the emphasis toward other ways of labeling synthetic oligonucleotides. Although many different types of non-radioactive labels have found their use in biological detection assays, use of fluorescent labels has expanded rapidly in recent years due to both improvements in detection instrumentation and to the increased number of novel fluorescent labeling reagents.
The sensitivity and accuracy of fluorescence detection techniques are dependent on the physical and chemical characteristics of the dyes they employ. A common problem with many commercially available fluorescent labeling reagents is that they are not water-soluble and must be dissolved in organic solvents prior to substrate labeling in aqueous media. Such organic solvents can have a deleterious effect upon sensitive substrates. Another problem related to the dye""s chemical structure is non-specific staining of cellular matter by the dye, which reduces signal to noise ratio during observation.
One of the major issues related to fluorescent labeling of oligonucleotides is the availability of fluorescent dyes in one or another chemical form. Ideally, the chosen fluorescent dye would be available as a fully protected, modified phosphoramidite for direct labeling during DNA synthesis. However, phosphoramidites are substantially more expensive and less stable than their standard, unmodified counterparts. Consequently, an indirect labeling method is used when the chosen fluorescent dye is not available as a modified phosphoramidite. The indirect method requires reacting an activated fluorescent dye with a nucleotide or oligonucleotide into which a primary amino group has been incorporated, usually at the 5xe2x80x2 end. Therefore, a selectivity towards nitrogen nucleophiles is a highly desirable property of fluorescent dyes employed in indirect labeling of oligonucleotides.
Cyanine and related dyes offer many advantages over existing fluorescent labeling reagents. These dyes strongly absorb fluorescent light. Many cyanine and related dyes are relatively photo-stable and do not rapidly bleach under the fluorescence microscope. They can be covalently attached to biological and non-biological markers to make these materials fluorescent. By synthesizing structural modifications of the chromophore portion of cyanine dyes, different fluorescent labeling reagents absorbing and emitting in a broad spectrum range from 400 to nearly 1100 nm can be obtained. Thus, reactive derivatives of these dyes can be made for assays that require simultaneous measurement of a number of labeled materials. Additionally, due to their relatively small size, cyanine dyes minimally perturb the function of the labeled product. Finally, the versatility of functional groups that can be incorporated into cyanine dyes permits control over the solubility of the dye and labeled product and helps reduce non-specific binding of the labeled materials to irrelevant components in an assay mixture (Waggoner, U.S. Pat. No. 5,569,587 and U.S. Pat. No. 5,627,027).
In order to improve covalent attachment of cyanine dyes to target molecules, techniques for activating cyanine dyes by the incorporation of a reactive functional group (or activating group) have been developed. Waggoner (U.S. Pat. No. 5,569,587 and U.S. Pat. No. 5,627,027) has presented numerous cyanine dye derivatives that can be used as covalently reacting molecules. The reactive groups used in these dyes are isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono-or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, aziridine, sulfonyl halide, acid halide (except for fluorides), hydroxysuccinimide ester, hydroxy sulfosuccinimide ester, imido ester, glyoxal and aldehyde. However, these types of cyanine dyes exhibit little or no selectivity towards nitrogen nucleophiles over competing species such as water or hydroxyl groups and, therefore, are not effective in labeling amino-modified materials such as amino-oligonucleotides.
The incorporation of carboxylic groups into the basic cyanine structure to increase solubility of the dye in water and to permit fluorescent labeling through the use of derived active esters has been suggested by Waggoner (U.S. Pat. No. 4,981,977 and U.S. Pat. No. 5,627,027). Miraha et al. (U.S. Pat. No. 4,337,063) and Masuda et al. (U.S. Pat. No. 4,404,289; U.S. Pat. No. 4,405,711; U.S. Pat. No. 4,414,325) have synthesized a variety of cyanine dyes with carbodiimides, anhydrides, active esters and other activating groups. These patents show that these reagents can be used as photographic sensitizers. However, most of the dyes mentioned in those patents are only weakly fluorescent. They are not especially photostable, and their solubility properties are not optimal for many uses that would involve fluorescence detection of labeled materials. Moreover, these dyes also lack selectivity towards nitrogen nucleophiles.
In summary, existing chemistries for attaching cyanine dyes to nucleotides and oligonucleotides exhibit little or no selectivity towards nitrogen nucleophiles over competing species such as water or hydroxyl groups. In addition, the existing methodologies are generally complicated, expensive, and produce undesirable organic by-products. Therefore, a need exists for novel methods of activating fluorescent dyes which overcome the difficulties of the prior art. A need also exists for methods of labeling nucleotides and oligonucleotides in both organic and aqueous solvents.
The present invention is based on the discovery that an acyl fluoride group incorporated into a cyanine dye structure provides desirable selectivity towards nitrogen nucleophiles. Fluorine is a halogen with unique physical and chemical properties such as, for instance, its ability to react with some of the noble gasses. Similarly, fluorides have strikingly different properties from the other halides. Amino acid fluorides are known as effective reagents for peptide coupling due to their selective reactivity towards amino groups (Carpino et al., U.S. Pat. No. 5,360,928). Fluorides, however, have never been suggested by the prior art as a desirable activating group of cyanine dyes. It is a surprise discovery of the present invention that cyanine dyes activated by acyl fluoride groups have specific reactivity towards nitrogen nucleophiles compared to oxygen nucleophiles, while remaining stable in aqueous solutions under labeling conditions.
One aspect of the present invention provides an activated dye having the formula: 
wherein each dotted line represents carbon atoms necessary to form a fused substituted or unsubstituted aromatic ring; n is an integer selected from the group consisting of 1, 2 and 3; X and Y are selected from the group consisting of S, O, N, CH2, and C(CH3)2; at least one of said R1 and R2 comprises a sulfonic acid or sulfonate group attached to the aromatic ring; and R3 and R4 are independently selected from the group consisting of alkylcarboxylate, activated alkylcarboxylate and an inert group; wherein at least one of the R3 and R4 groups is alcylcarboxylate or activated alkylcarboxylate with carboxyl group converted to an acyl fluoride and wherein the inert group is a group that is inert towards acyl fluoride and has a sterical structure which allows aminoacylation of the acyl fluoride group. The inert group may be selected from the group consisting of alkanes, alkenes, dienes, alkynes, arenes, ketones, amides and ethers. When necessary to improve stability, the alkyl carbon adjacent to the acyl fluoride group may also be substituted with a substituent group that is neither reactive nor sterically hinders aminoacylation.
Particularly, the present invention relates to cyanine dyes with acyl fluoride(s) activating group(s). According to one embodiment of the present invention, the cyanine dye is selected from the group consisting of Cy5, benzyl Cy5, dibenzyl Cy5, Cy7 benzyl Cy7, and dibenzyl Cy7.
Another aspect of the present invention provides a method of activating a dye by substituting the hydroxyl of a carboxylic group of the dye with fluorine. The method includes the steps of:
(a) providing a dye having at least one carboxylic group;
(b) providing a fluorinating reagent capable of reacting with the carboxylic group of said dye to form acyl fluoride; and
(c) reacting said dye with said fluorinating agent under a condition sufficient for formation of at least one acyl fluoride group.
According to embodiments of the present invention, the dye can be a cyanine dye, and particularly Cy5, benzyl Cy5, dibenzyl Cy5, Cy7, benzyl Cy7 or dibenzyl Cy7. The fluorinating agent may be diethylaminosulfur trifluoride (DAST), cyanuric fluoride or tetramethylfluoro-formadinium hexafluorophosphate (TFFH) or any other fluorinating agent depending on the methodology used.
A further aspect of the present invention provides a method wherein luminescent dyes, which contain an acyl fluoride group, are used to label biological and non-biological materials at their amine site. The method includes the steps of
(a) providing an activated dye with at least one acyl fluoride group;
(b) providing a material containing a nitrogen nucleophile; and
(c) reacting said dye with said material under a condition sufficient to couple the dye to the material.
According to embodiments of the present invention, the dye can be a cyanine dye, and particularly Cy5, benzyl Cy5, dibenzyl Cy5, Cy7, benzyl Cy7 and dibenzyl Cy7. The materials may be polymers, DNA, drugs, toxins, cells, microbial materials, particles, proteins, amino-modified nucleic acids, combinations thereof and, particularly, amino-modified oligonucleotides. The dyes of this invention are advantageously soluble in an aqueous medium and allow a simple one-step labeling process.
The present invention provides both economic and technical advantages over the use of other activating groups in the labeling of biopolymers, and especially amino-modified oligonucleotides. The acyl fluoride group allows for fast reaction kinetics of cyanine dyes with target biopolymers which results in only a trace amount of hydrogen fluoride which is instantly neutralized by a reaction buffer. Thus, introduction of an acyl fluoride group into cyanine dyes simplifies and reduces the cost of their application and handling.